>50 AA (connected by peptide bonds) to be considered a protein,

can hydrolyze proteins. eg add 6 M HCl, reflux. heat solution to accelerate

reaction, condenser to prevent loss of product by evaporation. hydrolyze to
respective monomeric units (aas)

proteins: can be one polypeptide chain, or 2 or more

1 aa sequence
2 alpha-helix, beta-sheets
3 defined function; tetramer, etc. every monomer is a single polypeptide

proteins are amphoteric: can act as acid/base (electron acceptor/donor)

zwitterion: #positively chd grp = # negatively chd group. 0 net charge.
at different conditions, you can achieve zwitterionic form isoelectric pH
consider all ionizable sidechains when determining pI.

R groups det/dictates protein property. eg hydrophobic vs hydrophilic R

groups.
EXPT GOAL: isolate proteins, albumin (egg white) and casein (milk)

general steps: extraction, isolation, purification

diffn8:
extraction goal is to destroy cellular components / take it from intracellular
components.
disrupt cell membrane. cell membrane of plants, animals,
bacteria.
if from animals: expect CM to be soft. possible methods:
freezing and thawing, mechanical method (stirring, using mechanical
beater(?), s_?)
if from plants: expect rigid (w/ CW)
may use mortar and pestle, add sand and grind;
liquid N, freeze and pound

burst cell, release protein in extraction.

isolation: remove other cellular components.
2 methods: centrifugation, fractional precipitation (in expt).

centrifugation : basis of varying density. gravity has something to do w
density (lightwt sa taas supposedly). kinds of centrifuge. larger radius, larger
centrifugal force experienced
maximum speed, 10 mnutes etc. max out lang.

after centrifugation: fractional pption. 4 methods to form ppt.

1. isoelectric pption
 a. control pH of system. @pI, 0 net charge, and thus
insoluble in water. react w/ one another; aggregate/ppt
out. (solubility: same IMFA to be soluble)
2. thru temperature change
a. proteins clump together at colder temperatures.
deactivate protease (w/c cleave, denature proteins).
3. lowering of dielectric constant
a. add organic solvents (eg ethanol) to solution. polarity of
solution decreases. proteins are CHARGED
biomolecules; decrease polarity, decreased solubility.
EtOH is less polar than water. if u add more EtOH,
bababa polarity ng buong solution (reduce prot-water
intxn)
4. addition of salt
a. salt is ionic: instead of protein-water intxn, inaagawan
ng salt. salt-water intxn prevalent.

purification
traces of other components still present.
exptal: chromatographic techniques (separation techniques); SDS-PAGE (can
cut bands, melt gel);

SDS-PAGE
earlier: washed w destaining soln until clear, viewed in lightbox.

/ / / / // / / / / / / / / / / / / / /
protein of interest: albumin. produced in liver. when low body level of
albumin, indicates you have liver/kidney disease. liver disease: liver canno;;;
kidney: excrete albumin
albumin can be found in serum. serum is matrix of blood.
albumin uses: mainly for transpo of fats
add albumin to solution to stabilize enzyme? albumin interacts with
antibodies, so that the enzymes dont. first line of defense
pI of BSalbumin ~4.6, MW (BSA) 66.4 kDa

in expt: want egg white only. bc egg yolk is fatty. hassle iremove yolk.
albumin content is also higher in egg white anyway. then, added HOAc to
(disrupt cell mem), but mainly added bc we want to ppt out OTHER cellular
components with low pI. (lowers pH of solution).

next step: filtration thru cheesecloth. remove unwanted proteins. only

albumin in filtrate hopefully. add ammonium sulfate: salting out; trying to ppt
proteins thru salting out effect. add equiv vol of soln. ammonium sulfate,
why? cheap, high ionic character, effective disruptor of protein-water intxns.
discard ppt. then add ammonium sulfate again, centrifuge, then collect ppt.
kasi yung unang nag ppt yung others. 2 main proteins present in egg white:
globulins and albumins. at varying [salt]s iba ibang proteins nagp-ppt out.
some are more insoluble in dilute salt soln, some are more insol in
concentrated salt soln. technique used is salting in and salting
outttttttttt

salting in:
iin dilute solns, soluble / do not want to ppt [assumption]
salting in, papasok ng solution: you MAKE IT soluble.

salting out:
nilalabas yung protein, only when conc salt []. in general. there are
exceptions, though.
assume that globulins ppt out first [ explanation]

same [salt] added. pero bakit hindi same time nag ppt out?
saturated yun

salting in: trying to dissolve in to the protein ewan

casein: heterogenous mixture of proteins found in milk, phosphoprotein

produced in the mammary tissue; nutritionally-adequate protein bc essential
aas are present in it; pI 4.7

main > isoelectric pption. also : lowering of dielectric constant of H2O. oks
na pag ~pH 4,wash w ethanol > loweing o diel const . acetone more
volatile.

#####
isoelectric focusing is like SDS-PAGE: apply voltage based on proteins pI.
based on paper
presentation of data: yield of albumin, do not compare it to the casein.
statistical coeff: normalizeeeee (recording)

extr isol purifxn.

isol :albumin salting out
casen isoelectric pptation
E2 PROTEIN QUANTITATION THROUGH SPECTROPHOTOMETRIC METHODS

REaCTION W SUBSTRATESM ETCE TEC TECTERCETRCTE

confirm if may proteins nga na nakuha.

spectrophotome Use absorption of cpd of light; absorbance is directly

tric: prop to concentration: Beer-Lamberts Law. A = epsilon *
b *c
Applicable only when compound is capable of absorbing
light in the UV-VIS region
Properties of The diversity of protein structure must be considered in
proteins choosing the most suitable protein quantitation method.

- GLOBULAR PROTEINS
- eg albumin, antibodies.
- soluble in dilute salt soln

- FIBROUS PROTEIN
- collagen, keratin
- typically insoluble (in salt solns) unless hydrolyzed

- CONJUGATED PROTEINS
- Hemoglobin. proteins w non-aa components eg
glycoprotein, lipoprotein. prosthetic group. (iron
ligand = heme)
Casein is conjugated because it has phosphate group.
Basis for PHYSICAL PROPS
quantitative - presence of aromatic aas w/c absorbs EM rad
measurements maximally at 280 nm

CHEMICAL PROPS (react to make PRODUCTS which

absorb)
- dye binding to hydrophobic regions [eg Coomassie
 Brilliant Blue]
- peptide bond forming violet-colored complex with
Cu2+ [Biuret]
- reaction with oxidizing agents of Tyr and Trp to
phosphotungstic and phosphomolybdic acids
[BCA?]
ASSAYS IN THE BRADFORD ASSAY
EXPT - Bradford reagent (Coomassie Brilliant Blue G250 in
95% ethanol and 85% H3PO4)
- standard calib curve is constructed using BSA as
the std protein
like typical uv-vis. std calib curve, then conduct analysis
of sample. have known, fit in unknown.
(absorbance shift 465 nm 595 nm) (read at 595)
ADVANTAGES: simple, sensitive (up to 5 micro g / micro
L), few interferences
Interference: siya mismo mag aabsorb, or
aagawan yung complex to disassemble complex
ganern

Disadv: formation of dye-protein complex can be

affected by the number of basic amino acids.
Reading changes in basic aas. false result pag
super basic na kasi di kakabit coomassie blue.

BIURET ASSAY
- Biuret reagent: NaOH, hydrated copper (II) sulfate
- rxn bet Cu2+ in alkaline soln and 2 adjacent
peptide bonds

pinakamahalaga ang Cu2+. it binds to peptide bonds.

peptide bonds chelated by Cu2+, which is then reduced
to Cu+
@540 nm
Cu2+ Cu+. reduced. cannot retain +2ness.

ADVANTAGE : not affected by AAcomposition; detects

peptide bond easily
DISADV: lacks sensitivity. cannot read when less than 1
mg/mL ang sample.
Other Lowry
quantitation - combo of Biuret assay and [O] of tyr and trp w
methods Folin and Ciocalteaus reagent
- more sensitive than Biuret

Bicinchonic
- similar to Biuret, chelation of bicinchoninic acid to
Cu+
- first, proteins reduce Cu2+ to +. bicinchoninic
kakabit sa Cu+.
- most sensitive assay :)

Ninhydrin
- dets free amino nitrogen
- ninhydrin is yellow in color, reacts with N
producing deep purple soln
kung may free amino groups, dun kakabit yung
ninhydrin: Lys, Arg (guanidinium grp: N=N-N), terminal
NH3+. these are susceptible to ninhydrin rxn.

Kjeldahl
- measures total nitrogen in a sample
very common. used in food companies to det prot
contents. melamine scandal!reported as high prot
when really just high melamine (wc is N rich)
cleave N bonds. protein gagawing ammonium ion
ammonia. then titrate to get %N.

disadvantage: attributes all N% to the amino group,

but not the side chain. not accurate. small
discrepancy usually

diff proteins have diff hydrophobic regions.. how do

you know if thats really the protein? ganern.
standard. to correct for side chains

assumption: sample is similar in structure to std